JP2012508014A - CRHR2 peptide agonist and use thereof - Google Patents

Abstract

  The present invention is a selective corticotropin releasing hormone receptor type 2 (CRHR2) agonist and composition thereof for the treatment, amelioration or inhibition of cardiovascular conditions including but not limited to heart failure, It relates to new peptides. This new peptide agonist preferably includes modifications including PEGylated peptides. The invention further relates to methods for the treatment and prevention of diseases or disorders associated with CRHR2 activity.

Description

(Cross-reference of related applications)
This application claims the benefit of US Provisional Patent Application Nos. 61 / 111,233 (filed Nov. 4, 2008) and 61 / 178,890 (filed May 15, 2009).

(Field of Invention)
The present invention generally includes peptides useful as stress copine mimetics and constructs for peptide delivery, for the treatment of medical indications mediated by corticotropin releasing hormone receptor 2 activity. The present invention relates to pharmaceutical compositions and methods for treating a subject diagnosed with a disease, disorder or medical condition mediated by corticotropin releasing hormone receptor 2.

  Heart failure is a common cardiovascular disease, reaching a large proportion of the disease in the United States and Europe (Remme et al., Eur. Heart J., 2001, vol. 22, pp. 1527-1560). There are nearly 1 million hospitalized patients with acute heart failure each year in the United States alone. Currently, the readmission rate and mortality within 60 days after discharge have reached 30% to 40% (Cuffee et al., JAMA, 2002, vol. 287 (12), pp. 1541-7). In acute heart failure, impaired hemodynamic function, particularly very high left ventricular end-diastolic pressure, is common.

  Current treatment of acute heart failure consists of many factors and often varies from patient to patient. Diuretics, vasodilators, and cardiotonics remain central in the treatment of patients with acute heart failure, but these treatments are associated with mortality and high readmission rates.

  Furthermore, existing cardiotonic therapies (eg, dobutamine) provide improved cardiac output, but with increased heart rate and increased myocardial oxygen consumption. These cardiotonics also provide the potential for arrhythmia induction in heart failure patients. This cardiac trend is thought to be related to the energy expenditure and calcium drive associated with the direct cardiotonic effects of these drugs.

  A number of new approaches have been studied to address this growing unmet medical need, but with limited success in safely improving hemodynamic status and outcome in patients with this syndrome. Absent. One such drug, the peptide human urocortin 2 (h-UCN2), has been studied in healthy subjects and patients with heart failure. This peptide has been shown to increase left ventricular ejection fraction (LVEF) and cardiac output (CO) in a sheep heart failure model (Rademaker et al., Circulation, 2005, vol. 112, pp. 3624- 3624). 8 healthy subjects (Davis et al., J. Am. Coll. Cardiol., 2007, vol. 49, pp. 461-471) and 8 subjects with heart failure (Davis et al., Eur. Heart J., 2007) , Vol. 28, pp. 2589-2597) In subsequent studies of intravenous injection in LVEF and CO increased heart rate and decreased blood pressure at both doses examined in each of the two studies. Was accompanied. One hour intravenous injection of h-UCN2 for healthy subjects and patients appears to have been well tolerated.

  Human stress copine (h-SCP), a 40 amino acid peptide, is associated with h-UCN2, both of which belong to the corticotropin releasing hormone (CRH) peptide family. The biological effects of the CRH peptides are manifested by two 7-transmembrane G protein coupled receptors, CRH receptor type 1 (CRHR1) and CRH receptor type 2 (CRHR2). Although these receptors have a high sequence homology, different elements within the CRH peptide family can make significant differences in the relative binding affinity, the degree of receptor activation, and the selectivity of these two receptors. Show.

  Unlike many of the CRH group elements, h-SCP expresses greater selectivity for CRHR2 and acts as a mediator that assists in the process of weakening the initiation and maintenance of physiological stress (Bale et al., Nat. Genet. 2000, vol.24, pp.410-414; Kishimoto et al., Nat.Genet., 2000, vol.24, pp.415-419). In addition to its obvious role in physiological stress, h-SCP has been reported to manifest numerous other physiological effects. This is the endocrine system (Li et al., Endocrinology, 2003, vol. 144, pp. 3216-3224), central nervous system, cardiovascular system (Bale et al., Proc. Natl. Acad. Sci., 2004, vol. 101, pp. 3697-3702; Tang et al., Eur.Heart J., 2007, vol.28, pp. 2561-2562), pulmonary system, gastrointestinal system, renal system, skeletal muscle system, and inflammatory system (Moffatt et al., FASEB J. et al. , 2006, vol. 20, pp. 1877-1879).

  In addition, CRHR2 activity is associated with skeletal muscle consumption disorders such as sarcopenia (Hinkle et al., Endocrinology, 2003, vol. 144 (11), pp. 4939-4946), exercise activity and food intake (Ohata et al., Peptides, 2004, vol. 25, pp. 1703-1709) and contribute to the role of cardioprotection (Brar et al., Endocrinology, 2004, vol. 145 (1), pp. 24-35), and bronchial relaxation and It exhibits anti-inflammatory activity (Moffatt et al., FASEB J. 2006, vol. 20, pp. E1181 to E1187).

  PEGylation is the process of attaching one or more polyethylene glycol (PEG) polymers to a molecule. PEGylation processes are often applied to antibodies, peptides and proteins to improve their biopharmacological properties, sensitivity of compounds to proteolytic enzymes, short circulation half-life, short shelf life, low solubility, rapid renal clearance , And overcoming the potential for antibody generation against the administered drug (Harris et al., Nature, 2003, vol. 2, pp. 214-221; Hamidi et al., Drug Delivery, 2006, 3, pp. 399-409). Bailon et al., PSTT, 1998, vol.1 (8), pp.352356). Recently, the FDA has approved PEG polymers for use as excipients or substrates for food, cosmetics, and drugs. Overall, PEG polymers are relatively non-immunogenic, have little toxicity, and are excreted intact by the kidneys or in feces. These properties can provide numerous clinical benefits for a compound when this process is developed to preserve or improve the affinity, efficacy, and pharmacological profile of the parent molecule.

  The present invention is directed to general and preferred embodiments, each defined by the independent and dependent claims appended hereto, and is incorporated herein by reference. Preferred and representative features of the present invention will become apparent from the following Detailed Description, with reference to the drawings.

In certain embodiments, the present invention is a peptide having agonist activity against adrenocorticotropic hormone releasing hormone receptor type 2 (CRHR2) comprising the following amino acid sequence:
LSLDV PTNIM NLLFN IAKAK NLRAQ AAANA HLMAQ I,
Wherein at least one amino acid of the peptide is replaced with X, provided that this substitution is not made at positions 3, 29, and 33 of the amino acid sequence and where X is cysteine, tyrosine, or glutamic acid; or It relates to these pharmaceutically acceptable salts or amides.

  The CRHR2 peptide agonist embodied in the present invention comprises an amino acid sequence that is relatively similar to the sequences of human stress copine and human urocortin III.

  In one embodiment, the amino acid substitutions to the above referenced amino acid sequence are: X at position 1 instead of L; X at position 2 instead of S; X at position 4 instead of D; X; Position 6 X instead of P; Position 7 X instead of T; Position 8 X instead of N; Position 9 X instead of I; Position 10 X instead of M; Position 11 X instead of N; X instead of L at position 12; X instead of L at position 13; X instead of F at position 14; X instead of N at position 15; X instead of I at position 16 X at position 17 instead of A; X at position 18 instead of K; X at position 19 instead of A; X at position 20 instead of K; X instead of N at position 21; X instead of L at position 22; X instead of R at position 23; X instead of A at position 24; X instead of Q at position 25; X instead of A at position 27; X instead of A at position 28; X instead of A at position 30; X instead of H at position 31; X instead of L at position 32; X; position Selected from the group consisting of X instead of A at 34; X instead of Q at position 35; and X instead of I at position 36.

  The present invention also relates to pharmaceutically acceptable salts or amides of this CRHR2 peptide agonist, and is further directed to pharmaceutical compositions of this peptide in combination with one or more pharmaceutically acceptable excipients.

In yet another embodiment, the amino acid sequence of the CRHR2 peptide agonist is
XLSLD VPTNI MNLLF NIAKA KNLRA QAAAN AHLMA QI-NH ₂ ;
XTLSL DVPTN IMNLL FNIAK AKNLR AQAAA NAHLM AQI-NH ₂ ;
XFTLS LDVPT NIMNL LFNIA KAKNL RAQAA ANAHL MAQI-NH ₂ ; and XKFTL SLDVP TNIMN LLFNI AKAKN LRAQA ANAH LMAQ-NH ₂ .

  In another embodiment, the complex comprises the amino acid sequence and a linker attached to X of the CRHR2 peptide agonist. Preferably this X is cysteine. Preferably, this linker is acetamide or N-ethylsuccinimide.

  In yet another embodiment, the agonist peptide conjugate comprises polyethylene glycol (PEG) attached to a linker having a molecular weight of 80 kDa or less. Preferably, the PEG has a molecular weight of either about 2 kDa, about 5 kDa, about 12 kDa, about 20 kDa, about 30 kDa, or about 40 kDa.

  The linker is for easier and selective attachment of PEG with respect to the position of the amino acid sequence relative to the peptide, while at the same time PEGylation of the peptide extends the half-life of the PEGylated peptide, thereby providing therapeutic therapeutics to the patient. Profit duration is extended. Thus, substitutions to the amino acid sequence of a CRHR2 peptide agonist preferably results in at least one type X amino acid in the sequence. This ensures that the PEGylation of the peptide is directed to at least one position in the sequence.

  In certain embodiments, the CRHR2 peptide agonist is SEQ ID NO: 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 39, 40, and 41.

  Another aspect of the present invention is a subject suffering from, or diagnosed with, a disease, disorder, or medical condition mediated by corticotropin releasing hormone receptor type 2 activity, which is a metabolic disease or heart failure. It aims at the treatment method of the test subject. This method includes administering to a subject in need of such treatment an effective amount of a CRHR2 peptide agonist embodied in the present invention.

  Additional embodiments and advantages of the invention will be apparent from the discussion, schemes, examples, and claims below.

Analytical HPLC traces of the peptide agonist with SEQ ID NO: 102 derivatized with iodoacetamide-PEG after 2 hours reaction time and purification, respectively. Analytical HPLC traces of the peptide agonist with SEQ ID NO: 102 derivatized with iodoacetamide-PEG after 2 hours reaction time and purification, respectively. Mass spectrum graph of a peptide agonist with SEQ ID NO: 102 derivatized with iodoacetamide-PEG. The figure which shows the agonist effectiveness and selectivity of a peptide agonist with respect to human CRHR1 and CRHR2. The figure which shows the influence of the competitive antagonism between the peptide agonist of sequence number 1, and anti- sorbazine-30 (sequence number 118). The figure which shows the agonist concentration-effect curve of the various peptide agonist obtained by measuring cAMP stimulation in the SK-N-MC cell transfected by h-CRHR2. In SK-N-MC cells transfected with h-CRHR2, in the absence and presence of 10 μM CRHR2 peptide agonist with the sequences of SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112, respectively. The figure which shows the h-SCP (sequence number 1) agonist concentration-effect curve measured through cAMP stimulation. FIG. 5 shows relaxation of pre-contracted isolated rat aorta by a peptide agonist with SEQ ID NO: 1 and SEQ ID NO: 115 (h-UCN2). FIG. 3 shows changes in heart rate, left ventricular pressure, and coronary perfusion pressure in a Langendorff-perfused rabbit heart in the presence of a peptide agonist with SEQ ID NO: 1 and a placebo control solvent.

  The present invention relates to new peptides which are CHRH2 peptide agonists and compositions thereof for the treatment, amelioration or inhibition of cardiovascular conditions including but not limited to heart failure and metabolic diseases.

  In one embodiment of the invention, a method of treating or ameliorating heart failure in a subject in need of treatment or amelioration of heart failure includes administering to the subject a therapeutically effective amount of at least one CRHR2 peptide agonist. It is.

  In certain embodiments, the CRHR2 peptide is a mammalian peptide, particularly a mouse, rat, guinea pig, rabbit, dog, cat, horse, cow, pig, or primate peptide, or a modification thereof. is there. Preferably the peptide is a modified human peptide. Examples of CRHR2 peptide agonists of the present invention are described in detail in the following sections.

  Another embodiment of the invention includes a reactive group covalently linked to a peptide agonist. This reactive group is selected so that it can form a stable covalent bond with a polymer or other chemical moiety that extends the circulating half-life of the peptide within the subject. In one embodiment, such polymers include polyethylene glycol (PEG) polymers that extend the duration of the peptide in the subject's circulation to elimination. In this form, the reactive group acts as a linker between the peptides by reacting one hand with one or more amino acids of the peptide and reacting the other with the polymer. In another embodiment, the reactive group is first conjugated with PEG and then forms a chemical bond with the peptide. In a preferred embodiment of the modified peptide, the linker group is succinimide, more specifically N-ethylsuccinimide, or acetamide. Further, the linker can be vinyl sulfone or orthopyridyl disulfide. Preferably, chemical modification is performed on the isolated peptide, for example to increase reaction efficiency.

  Linkers useful for coupling to the peptide and PEG moieties can provide minimal immunogenicity and toxicity to the host. Examples of such linkers are described in Bailon et al., PSTT, 1998, vol. 1 (8), pp. 352-356, or Roberts et al., 2002, Adv. Drug Del. Rev. , Vol. 54, pp. 459-476. For examples of suitable chemical moieties, particularly PEG and equivalent polymers, see Greenwald et al., 2003, Adv. Drug Del. Rev. , Vol. 55, pp. 217-250. For example, in other polymer systems, styrene-maleic anhydride neocalcinostatin copolymer, hydroxylpropylmethacrylamide copolymer, dextran, polyglutamic acid, hydroxylethyl starch, and polyaspartic acid are similar in delivery and pharmacokinetics to the PEG system. Can be used to achieve properties.

  In certain embodiments of the invention, the CRHR2 peptide agonist includes an amidated C-terminus. The modification procedure can be performed on the isolated purified peptide or, in the case of solid phase synthesis, during the synthesis procedure. The procedure is described in Ray et al. , Nature Biotech. 1993, vol. 11, pp. 64-70; Cottingham et al. , Nature Biotech. 2001, vol. 19, pp. 974-977; Walsh et al. , Nature Biotech. , Vol. 24, pp. 1241-1252; and U.S. Patent Publication No. 2008/0167231.

  In one particular embodiment of the invention, the compound comprises a peptide of the amino acid sequence shown in SEQ ID NO: 82 or SEQ ID NO: 102 containing CONH2 at the carboxy terminus, together with a linker that is N-ethylsuccinimide or acetamide, Includes a linker attached to a cysteine residue at position 28, as well as a linker attached to a PEG polymer of about 20 kDa.

  Furthermore, one embodiment of the present invention provides a subject with a therapeutically effective amount of at least one CRHR2 peptide agonist of a subject suffering from or diagnosed with a disease, disorder or condition mediated by CHRH2 activity. It features a method of treatment comprising administering.

  Another embodiment of the invention further features a method of treating or inhibiting one or more CHRH2-mediated symptoms, illnesses, or progression of a disease, the method comprising at least one CRHR2 peptide agonist agent It includes administering an effective amount to a patient in need of treatment.

  In a further embodiment of the invention, there is provided a process for producing a pharmaceutical composition comprising mixing any CRHR2 peptide agonist and a pharmaceutically acceptable carrier.

Terms and Definitions The invention is best understood by referring to the following definitions, figures, and representative disclosure provided herein.

The following abbreviations are sometimes used herein: pA ₅₀ or pEC ₅₀ = the negative logarithm, base 10, of the agonist concentration required to produce half the maximum effect; SEM = standard error; Log DR = logarithm with base 10 agonist dose ratio; MW = molecular weight; cAMP = adenosine 3 ′, 5′-cyclic monophosphate; cDNA = complementary DNA; kb = kilobase (1000 base pairs) KDa = kilodalton; ATP = adenosine 5'-triphosphate; nt = nucleotide; bp = base pair; PAGE = polyacrylamide gel electrophoresis; PCR = polymerase chain reaction; nm = nanomolar.

  The terms “comprising”, “containing”, and “including” are used herein in their open, non-limiting sense.

  “Administration” or “administering” means providing a drug to a patient in a pharmacologically useful manner.

  A “composition” is obtained directly or indirectly from a product comprising a compound of the invention (eg, a product comprising a particular ingredient in a particular amount, and such a combination of a particular ingredient in a particular amount. Means any product).

  “Compound” or “drug” means a CRHR2 peptide agonist or a pharmaceutically acceptable form thereof. “Conjugate” means a chemical compound formed by the joining of two or more compounds.

  “Dosage form” means one or more compounds in a vehicle, carrier, excipient, or device suitable for administration to a patient. “Oral dosage form” means a dosage form suitable for oral administration.

  “Dose” means a unit of drug. Usually, a dose is supplied as a single dosage form. The dose can be administered to the patient according to various dosage regimens. Common dosage regimes include once daily oral (qd), twice daily oral (bid), and three times daily oral (tid).

  “Form” means various isomers and mixtures of one or more CRHR2 peptide agonists. The term “isomer” refers to compounds that have the same composition and molecular weight but differ in physical and / or chemical properties. Such materials have the same number and kind of atoms but different structures. The structural difference can be a structural difference (geometric isomer) or a polarization plane rotation difference (stereoisomer). The term “stereoisomer” refers to isomers that have the same configuration but differ in the arrangement of their atoms in space. Enantiomers and diastereomers are stereoisomers in which the asymmetrically substituted carbon atom acts as a chiral center. The term “chiral” refers to a molecule that does not overlap the mirror image, which means that there is no axis of symmetry and no plane or center.

  “Medicament” means a product for use in a subject in need thereof for the prevention, treatment or amelioration of drug-related diseases such as drug addiction, drug abuse or drug-induced diseases.

  “Patient” or “subject” means an animal, preferably a mammal, more preferably a human, in need of therapeutic intervention.

  “Pharmaceutically acceptable” means a molecular entity or composition of sufficient purity and quality for use in the composition or pharmaceutical formulation of the present invention. Since both human use (clinical and commercial) and veterinary use are equally included within the scope of the present invention, the formulation includes any composition or drug for human or veterinary use. Will be included.

  “Pharmaceutically acceptable excipient” is used as an excipient, carrier or diluent that is added to a pharmacological composition or that facilitates administration of a drug and is compatible with the drug, A substance that is not toxic, biologically acceptable, or biologically suitable for administration to a patient, such as an inert substance. Examples of excipients include calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

  “Pharmaceutically acceptable salts” have sufficient purity and quality for use in the compositions or pharmaceutical formulations of the present invention, are well tolerated for use in pharmaceuticals, and are sufficiently non-toxic. Is an acid salt or base salt of a compound of the invention. Suitable pharmaceutically acceptable salts include acid addition salts, which include, for example, drug compounds such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carboxylic acid. Alternatively, it can be formed by reaction with a suitable pharmaceutically acceptable acid such as phosphoric acid.

“CRHR2 peptide agonist” means a peptide or derivative thereof that exhibits agonistic activity against corticotropin releasing hormone receptor type 2 (CRHR2). Preferably, the CRHR2 peptide agonist is a stress copine derivative or variant that may also exhibit activity against adrenocorticotropic hormone releasing hormone receptor type 1 (CRHR1). A CRHR2 peptide agonist is generally a selective CRHR2 agonist with relatively low activity against CRHR1. Selectivity for a receptor, as used herein, is that a peptide is selective compared to other receptors (the peptide can also induce activity against it but is less effective). It refers to the effectiveness of the peptide to induce an active response to the receptor. The definition of CRHR2 peptide agonist is not limited to agonists, and may include partial agonists. The CRHR1 and CRHR2 activities of CRHR2 peptide agonists can be assessed, for example, with an adenosine 3 ′, 5′-cyclic monophosphate (cAMP) assay.
Very similar to the CRHR2 activity of stress copine (h-SCP),

  A “therapeutically effective amount” is within a tissue system, animal or human, including therapeutic alleviation of the symptoms of the disease or disorder being treated or prevented as sought by a researcher, veterinarian, physician or other clinician. Means the amount of a compound that manifests a biological or pharmaceutical response.

  As used herein, the term “treating”, unless stated otherwise, reverses the disease or condition to which the term applies, or one or more symptoms of such a disease or condition. Means to alleviate, inhibit or prevent progression. As used herein, the term “treatment” refers to the act of treating, unless otherwise stated.

Compound The present invention relates to the following peptides and modified products thereof. The compounds of the present invention further include novel and selective CRHR2 agonist peptides and modifications thereof.

Furthermore, the compounds of the invention refer to peptide moieties that bind to CRHR2 or complex with CRHR2, such as h-SCP or pseudo-h-SCP peptides. Preferred compounds are, for example, measured at a pA ₅₀ in the range of about 7.5 or higher in the cAMP assay, or pK _I (negative logarithm of K _I in the range of about 7.5 or higher in the radioligand binding assay. ), And is a peptide having agonist activity against CRHR2. In addition to showing binding affinity, a CRHR2 peptide agonist should show some degree of receptor activation. Thus, peptides homologous to h-SCP are preferred because they naturally have similar physical and chemical properties.

  Those belonging to the group of adrenocorticotropic hormone-releasing factors exhibit a moderately short half-life. CRHR2 peptide agonists are expected to provide unique therapeutic properties. For the treatment of diseases mediated by CRHR2, including but not limited to cardiovascular and metabolic diseases, one embodiment of the present invention is directed to long acting forms of peptide agonists. Long-acting CRHR2 peptide agonists are particularly beneficial when treating chronic diseases that require continuous therapeutic exposure and when there is a problem with patient compliance with prescription drug treatment.

  Accordingly, one embodiment of the present invention generally provides h-SCP sequence changes, site-specific sequence changes, and spatial or spatial, such that desirable therapeutic properties and / or structure-activity relationships for CRHR2 are retained. The purpose is to consider three-dimensional interference.

  Examples of CRHR2 peptide agonists are optionally amidated at the C-terminus, and these are shown in Tables 1-5. The reactive group or linker is preferably succinimide or acetamide. The modified peptide optionally contains a PEG group. The PEG varies in length and weight, and is preferably about 20 kDa. If desired, the number of reactive groups may be greater than one, with one reactive group being preferred.

^aＮＥＳ−ＰＥＧ−ＮＥＭ：ＰＥＧ鎖の非連結末端に、Ｎ−エチルマレイミドキャップを備えた、ダブル末端の直鎖状ＰＥＧ。

^a NES-PEG-NEM: Double-terminal linear PEG with an N-ethylmaleimide cap at the unlinked end of the PEG chain.

  The pharmaceutical compounds of the present invention also include a mixture of stereoisomers or the respective pure isomers or substantially pure isomers. For example, the compound may optionally have one or more asymmetric centers on the carbon atom containing any one substituent. Thus, this compound may exist in the form of an enantiomer or diastereomer or a mixture thereof. When the compound contains a double bond, the compound may exist in the form of geometric isomerism (cis-compound, trans-compound), and when the compound contains an unsaturated bond such as carbonyl, The compounds may exist in tautomeric forms and the compounds also include these isomers or mixtures thereof. Starting compounds in the form of racemic mixtures, enantiomers or diastereomers may be used in the processes for preparing the compounds. When the present compounds are obtained in diastereomeric or enantiomeric forms, they can be separated by conventional methods such as chromatography or fractional crystallization. In addition, the present compounds include their inner salts, hydrates, solvates or isogenic.

  Furthermore, suitable pharmaceutical compounds are those that exert local physiological or systemic effects after penetrating the mucosa or, in the case of oral administration, after being transported with saliva to the gastrointestinal tract. The dosage forms prepared from the formulations according to the invention are particularly suitable for drug compounds that provide activity over a long period of time, especially drugs that have a half-life of at least several hours.

Synthetic Routes and Purification An “isolated” peptide is substantially free of cellular material or separated from cellular material, or derived from the cell or tissue source from which the peptide was produced and isolated Is separated from other contaminating proteins or is substantially free of chemical precursors or other chemicals when the peptide is chemically synthesized. For example, a protein that is substantially free of cellular material is less than about 30%, or preferably less than 20%, or more preferably less than 10%, or even more preferably less than 5%, or even more contaminated protein by dry weight. Protein preparations may be included, preferably having less than 1%.

  In preferred embodiments, the isolated peptide is substantially pure. Thus, if the peptide is produced recombinantly, it is substantially free of media, such as less than about 20%, or more preferably less than 10%, or even more preferably less than 5% of the protein preparation volume, or More preferably, it contains less than 1% medium. If the protein is produced by chemical synthesis, it is substantially free of chemical precursors or other chemicals, i.e. separated from the chemical precursors or other chemicals involved in the synthesis of the protein. Yes. Thus, such a preparation of a peptide comprises less than about 30%, or preferably less than 20%, or more preferably less than 10%, or more preferably 5% of a chemical precursor or compound other than the peptide of interest by dry weight. %, Or more preferably less than 1%.

Recombinant production Peptide expression in the cellular environment can be achieved by the use of isolated polynucleotides. An “isolated” polynucleotide is one that is substantially separated from nucleic acid molecules with different nucleic acid sequences or does not include nucleic acid molecules with different nucleic acid sequences. Isolated polynucleotide molecule embodiments include cDNA, genomic DNA, RNA, and antisense RNA. Preferred polynucleotides are obtained from biological samples derived from humans, such as from tissue samples.

  Vectors can be used to supply and propagate polynucleotides encoding the peptide of SEQ ID NO: 1. Introduction of such a vector into a host cell can result in production of mimetic stress copine encoded mRNA or protein. Another method is to combine the expression vector with purification elements including, but not limited to, transcription factors, RNA polymerase, ribosomes, and amino acids to produce an effective transcription / translation reaction in cell-free conditions. Can do. The mimetic stress copine peptide expressed from the resulting reaction can be isolated for further purification, modification, and / or formulation.

  The term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A plasmid, which is a typical example of a vector, refers to a circular double stranded DNA loop into which additional DNA segments can be inserted. Another example of a vector is a viral vector into which additional DNA segments can be inserted. Certain vectors are capable of self-replication in host cells into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, and are thereby replicated along the host genome. Furthermore, specific vectors (expression vectors) can direct the expression of genes to which they are operably linked. Useful vectors in recombinant DNA techniques can be in the form of plasmids. Alternatively, other forms of vectors that provide equivalent functions, such as viral vectors (eg, replication defective retroviruses, adenoviruses, and adeno-associated viruses) will be selected by those skilled in the art to suit the intended use. Can do.

  A host cell refers to a cell that contains a DNA molecule that is on a vector or integrated into a cell chromosome. The host cell can be either a native host cell that contains the DNA molecule endogenously or a recombinant host cell. An example of a host cell is a recombinant host cell, which is a cell transformed or transfected with a foreign DNA sequence. When such foreign DNA is introduced inside the cell membrane, the cells are transformed with the foreign DNA. The foreign DNA may or may not be incorporated (covalently linked) into the chromosomal DNA constituting the cell's genome. For example, in prokaryotes and yeast, foreign DNA can be retained on episomal elements such as plasmids. For eukaryotic cells, a stably transformed or transfected cell is one in which foreign DNA is integrated into the chromosome, which is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of eukaryotic cells to establish cell lines or clones consisting of a population of daughter cells containing exogenous DNA. A clone refers to a group of cells derived from one cell or common ancestor by mitosis. A cell line refers to a clone of a primary cell that can be stably grown in vitro for many generations. Recombinant host cells include bacteria such as E. coli, fungal cells such as yeast, mammalian cells such as human, bovine, porcine, monkey and rodent cell lines, cell lines derived from Drosophila and silkworms. It can be prokaryotic or eukaryotic, including insect cells. Recombinant host cell refers not only to a particular subject cell, but also to the progeny or potential progeny of such a cell. In particular, such progeny may not be identical to the parental cell, but are still included within the terminology, as certain modifications may occur in later generations, either due to mutations or environmental effects Is intended.

  Exemplary vectors of the invention include expression specifically designed to allow DNA to be exchanged between hosts, such as bacteria-yeast or bacteria-animal cells, or bacteria-fungal cells, or bacteria-invertebrate cells. Systems are also included. Many clone vectors are known to those skilled in the art, and the selection of an appropriate clone vector is within the discretion of those skilled in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells, see, for example, Maniatis et al. (1990), “Molecular Cloning: A Laboratory Manual,” vol. 2: See chapters 16 and 17 of 16.3 to 16.81.

  In order to achieve high levels of expression of a clonal gene or nucleic acid (eg, cDNA encoding a mimetic stress copine peptide), the nucleotide sequence corresponding to the mimetic stress copine peptide sequence is preferably a strong promoter that induces transcription, It is subcloned into an expression vector containing a transcription / translation terminator and (in the case of a nucleic acid encoding a protein) a ribosome binding site for translation initiation. Suitable bacterial promoters are known in the art, for example, Sambrook et al., (1989), MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbold, Cold Spring. Rev. 60 (3): 512-38. Bacterial expression systems for expressing mimetic stress copine proteins disclosed in the present invention can be used in, for example, E. coli, Bacillus, and Salmonella (Palva et al., 1983, Gene, 22: 229-235; Mosbach et al., 1983, Nature, 302: 543-545). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are known in the art and are also commercially available. In exemplary embodiments, the eukaryotic expression vector is a baculovirus vector, an adenovirus vector, an adeno-associated vector, or a retrovirus vector.

  A promoter refers to a DNA regulatory sequence involved in binding RNA polymerase and initiating transcription of a gene. The promoter is often upstream (ie 5 ') of the transcription start site of the gene. A gene refers to a DNA segment involved in the production of a peptide, peptide, or protein, including a coding region, a non-coding region before (5′UTR) and after (3′UTR) the coding region, and individual Non-coding sequences (introns) that intervene between the coding segments (exons). Coding refers to the use or termination signal of a particular amino acid in a three base triplet (codon) of DNA or mRNA.

  The promoter used to direct expression of the polynucleotide can be selected by routine procedures to suit a particular application. If desired, the promoter is arranged at a position where the distance from the transcription start site derived from a different species is approximately the same as the distance from the transcription start site under natural conditions. However, as will be apparent to those skilled in the art, some difference in this distance can be tolerated without loss of promoter function.

  In addition to the promoter, the expression vector may contain a transcription unit or expression cassette that contains all the additional elements required for the expression of the polynucleotide encoding the mimetic stress copine in the host cell. A typical expression cassette contains a promoter operably linked to a polynucleotide sequence encoding a mimetic stress copine peptide, and is required for efficient polyadenylation of transcription, ribosome binding sites, and translation termination Signal is contained. The polynucleotide sequence encoding the canine mimetic stress copine peptide can be linked to a cleavable signal peptide sequence that facilitates secretion of the encoded protein by the transfected cell. Representative signal peptides include signal peptides from tissue plasminogen activator, insulin, and neuron growth factor, and heliothis viruses juvenile hormone esterase. Additional elements of this cassette may include enhancers and introns with functional splice donor and acceptor sites if genomic DNA is used as the structural gene.

  In addition to the promoter sequence, the expression cassette may further include a transcription termination site downstream of the structural gene provided for efficient termination. The termination site can be obtained from the same gene as the promoter sequence, the human stress copine gene, or another gene.

  In an exemplary embodiment, any vector suitable for expression in eukaryotic or prokaryotic cells known in the art can be used. Representative bacterial expression vectors include plasmids such as pBR322 series plasmids, pSKF, pET23D, and fusion expression systems such as GST and LacZ. Examples of mammalian expression vectors include pCDM8 (Seed, 1987, Nature, 329: 840) and pMT2PC (Kaufman et al., 1987, EMBO J., 6: 187-195). Commercially available mammalian expression vectors that may be suitable for recombinant expression of the peptides of the invention include, for example, pMAMneo (Clontech (Mountain View, CA)), pcDNA4 (Invitrogen (Carlsbad, Calif.)), PCiNeo (Promega). (Madison, WI)), pMC1neo (Stratagene (La Jolla, CA)), pXT1 (Stratagene (La Jolla, CA)), pSG5 (Stratagene (La Jolla, CA)), EBO-pSV2-neo (ATCC 37593) pBPV-1 (8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199) PRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and lZD35 (ATCC 37565).

  In order to provide a convenient method of isolation, an epitope tag can also be added to the recombinant protein, for example c-myc, hemoglutinin (HA) tag, 6-His tag, maltose binding protein, VSV-G. There are tags, or anti-FLAG tags, and others available in the art.

  Expression vectors containing control elements from eukaryotic viruses can be used in eukaryotic expression vectors such as vectors derived from SV40 vectors, papillomavirus vectors, and Epstein-Barr viruses. Other representative eukaryotic vectors include pMSG, pAV009 / A +, pMTO10 / A +, pMAMneo 5, baculovirus pDSVE, and other CMV promoters, SV40 early promoter, SV40 late promoter, metallothionein promoter, mouse murine breast Examples include tumor virus promoters, rous sarcoma virus promoters, polyhedrin promoters, and other vectors that allow protein expression under the induction of other promoters that are effective in expression in eukaryotic cells.

  Some expression systems have markers that provide gene amplification, such as neomycin, thymidine kinase, hygromycin B phosphotransferase, dihydrofolate reductase. As another method, a high-yield expression system that does not use gene amplification is also suitable. For example, a sequence encoding a mimic stress copine peptide under the induction of a polyhedrin promoter or other strong baculovirus promoter is used. Use of the baculovirus vector in insect cells provided.

  The elements that can be included in the expression vector further include a replicon that functions in E. coli, a gene encoding antibiotic resistance to allow selection of bacteria harboring the recombinant plasmid, and controlled insertion of eukaryotic sequences. Also included are unique restriction sites in the non-major region of the plasmid that allow A particular antibiotic resistance gene can be selected from a number of resistance genes known in the art. Prokaryotic sequences can be selected so that they do not interfere with eukaryotic DNA replication as needed or desired.

  Known transfection methods can be used to produce bacterial, mammalian, yeast or insect cell lines that express large amounts of SCP mimetics. This SCP mimetic is then purified using standard techniques such as selective precipitation with substances such as ammonium sulfate, column chromatography, and immunopurification.

  Transformation of eukaryotic cells and prokaryotic cells can be performed by standard techniques (eg Morrison, 1977, J Bact., 132: 349-351; Clark-Curtis & Curtis, Methods in Enzymology, 101: 347-362). See).

  Any known technique suitable for introducing a heterologous nucleotide sequence into a host cell can be used to introduce the expression vector. This includes the use of reagents such as Superfect (Qiagen), liposomes, calcium phosphate transfection, polybrene, protoplast fusion, electroporation, microinjection, plasmid vectors, virus vectors, gene gun particle acceleration (gene gun), Or any other known method for introducing cloned genomic DNA, cDNA, synthetic DNA or other heterologous genetic material into a host cell (see, eg, Sambrook et al., Supra). The particular genetic engineering procedure chosen should be capable of successfully introducing at least one gene into a host cell capable of expressing mimic stress copine RNA, mRNA, cDNA, or gene.

  As will be apparent to those skilled in the art, for stable transfection of mammalian cells, depending on the expression vector and transfection technique used, only a small number of cells will have heterologous DNA in their genome. Can be integrated. In order to identify and select these components, a gene that encodes a selectable marker (eg, resistance to antibiotics) can be introduced into the host cells along with the gene of interest. Representative selectable markers include those that confer resistance to drugs such as G-418, puromycin, geneticin, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be selected and identified by drug selection (eg, cells incorporating the selectable marker gene will survive and other cells will die).

  By inserting a heterologous regulatory element into a stable cell line or clonal microorganism, for example, target homologous recombination (for example described in US Pat. No. 5,272,071 and PCT International Publication No. WO 91/06667). ) And the like can be operably linked to the endogenous gene to activate this endogenous gene expression. After introducing the expression vector into the cell, the transfected cell is preferably cultured under favorable conditions optimal for expression of the mimetic stress copine peptide, and mimic stress copin peptide from the culture using standard techniques described below. Is recovered. Methods for culturing prokaryotic or eukaryotic cells are known in the art and are described, for example, in Sambrook et al. (Supra); Freshney, 1993, CULTURE OF ANIMAL CELL, 3rd ed. checking ...

  As an alternative to using cell lines for peptide production, cell-free systems, prokaryotic cell systems (Zubay G., Annu Rev Genet., 1973, 7: 267-287) and eukaryotic cell systems ( Pelham et al., Eur J Biochem., 1976, 67: 247-256; Anderson et al., Meth Enzymol., 1983, 101: 635-644) have been shown to be possible. These systems can be utilized for either mRNA or DNA nucleotides for peptide synthesis reactions. In a preferred technique for cell-free peptide production, reticulocyte lysate, RNA polymerase, nucleotides, salts, and ribonuclease inhibitors are rapidly linked transcription / translation reactions (TNT®, Promega (Madison, Madison, WI, U.S.A.)).

Synthetic Production Peptides embodied in the present invention are described in Merrifield, J. et al. Am. Chem. Soc. 15: 2149-2154 (1963), and can be prepared using solid phase synthesis techniques. Other peptide synthesis techniques are described in, for example, M.M. Bodanzky et al. (1976) Peptide Synthesis, John Wiley & Sons, 2d Ed. Kent and Clark-Lewis, Synthetic Peptides in Biology and Medicine, p. 295-358, eds. Alitaro, K.M. Science Publishers, (Amsterdam, 1985); as well as other reference studies known to those skilled in the art. A summary of peptide synthesis techniques can be found in J. Stuart and J.M. D. Young, Solid Phase Peptide Synthelia, Pierce Chemical Company, Rockford, III. (1984), which is incorporated herein by reference. Solution synthesis of peptides can also be used, which is described in The Proteins, Vol. II, 3d Ed. , P. 105-237, Neuroth, H .; Et al., Academic Press, New York, N .; Y. (1976). Suitable protecting groups for use in such syntheses are described in the above references, as well as in J. Am. F. W. McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York, N .; Y. (1973), which is incorporated herein by reference. In general, these synthetic methods involve the sequential addition of one or more amino acid residues or suitable protected amino acid residues to a growing peptide chain. Usually, either the amino group or the carboxyl group of the first amino acid residue is protected by a suitable, selectively removable protecting group. Different, selectively removable protecting groups are utilized for amino acids containing reactive side groups such as lysine.

  The block synthesis method can also be applied to both the solid phase method and the solution method of peptide synthesis. Rather than sequentially adding a single amino acid residue, a preformed block containing two or more amino acid residues in sequence is used as the starting subunit or added sequentially Used as a unit (not a single amino acid residue).

  Solid phase synthesis is used as an example to attach a protected or derivatized amino acid to an inert solid support via an unprotected carboxyl or amino group. Next, the amino or carboxyl protecting group is selectively removed and the next amino acid in the sequence having a suitably protected complementary (amino or carboxyl) group is already attached to the solid support. React with the residue. Next, the amino or carboxyl protecting group is removed from the newly added amino acid residue, and the next amino acid (suitably protected) is added, followed by the same procedure. After all of the desired amino acids are linked in the appropriate sequence, the remaining terminal and side group protecting groups (and solid support) are removed sequentially or simultaneously to yield the final product peptide. The peptides of the present invention preferably do not contain benzylated or methylbenzylated amino groups. Such protecting group moieties can be used in the course of synthesis, but are removed before using the peptide. As described elsewhere, additional reactions that form intramolecular linkages to constrain conformation may be required.

  Solid support synthesis was performed using an automated protein synthesizer (Protemist (registered trademark), CellFree Sciences (Matsuyama, Ehime Prefecture, Japan 790-8777); Symphony SMPS-110, Rainin (Woburn, MA, USA). An ABI 433A peptide synthesizer, Applied Biosystems (Foster City, CA, USA)). These devices have the ability to perform automated protein reactions that allow greater control and optimization of synthesis.

Purification A number of procedures can be employed to isolate or purify the peptides of the present invention. For example, the peptide can be purified using column chromatography based on the physical properties (ie, hydrophobicity) of the peptide. Alternatively, proteins having established molecular adhesion properties can be reversibly fused to the peptides of the present invention. With the appropriate ligand of the fusion protein, the mimetic stress copine peptide is selectively adsorbed onto the purification column and then released from the column in a substantially pure form. The fusion protein can then be removed by enzymatic activity. Another column purification strategy can employ antibodies raised against mimetic stress copine peptides. These antibodies are bound to a column matrix and the peptide can be purified through this immunoaffinity column.

  The recombinant protein can be separated from the host reaction by a suitable separation technique such as salt fractionation. This method can be used to separate unwanted host cell proteins (or proteins from cell culture media) from the recombinant protein of interest. An example of a typical salt is ammonium sulfate, which precipitates the protein by effectively reducing the amount of water in the protein mixture (the protein precipitates based on its solubility). The more hydrophobic the protein, the more likely it will precipitate at lower ammonium sulfate concentrations. A typical isolation protocol involves adding saturated ammonium sulfate to the protein solution, resulting in an ammonium sulfate concentration between 20-30% and precipitating most hydrophobic proteins. Discard the precipitate (if the protein of interest is not hydrophobic) and add ammonium sulfate to the supernatant to a concentration known to precipitate the protein of interest. This precipitate is then dissolved in a buffer to remove excess salt to achieve the desired purity, for example by dialysis or membrane separation purification methods. Other known methods that depend on the solubility of the protein (eg, cold ethanol precipitation) can be used to fractionate complex protein mixtures.

  In other examples of isolation or purification techniques, the molecular weight of the peptides of the present invention is used to use ultrafiltration through membranes of various pore sizes (eg, Amicon or Millipore membranes) with dimensions larger or smaller than the protein of interest. It can be used to separate from other proteins. As a first step, the protein mixture is ultrafiltered through a membrane with a pore size having a molecular weight cut-off smaller than the molecular weight of the protein of interest. What remains from this ultrafiltration is then subjected to ultrafiltration with a membrane having a molecular cutoff greater than the molecular weight of the protein of interest. The recombinant protein passes through the membrane and into the filtrate, which can be passed through chromatography.

Chemical Modification Peptides of the present invention can be used to modify structural analogs of the peptide by chemical modifications such as maleimide capping, polyethylene glycol (PEG) linkage, maleidification, acylation, alkylation, esterification, and amidation. Can be a target for production. Those skilled in the art will appreciate that there are a variety of chemical modification techniques and moieties. For example, U.S. Pat. Nos. 5,554,728, 6,869,932, 6,828,401, 6,673,580, 6,552,170, See 6,420,339, U.S. Patent Publication No. 2006/0210526, and PCT International Publication No. WO 2006/136586. Preferably, chemical modification is performed on the isolated peptide, for example to increase reaction efficiency.

  In certain embodiments of the invention, the peptides of the invention include an amidated C-terminus. The peptide modification procedure can be performed on the isolated purified peptide or, in the case of solid phase synthesis, can be performed during the synthesis procedure. Such a procedure is described in Ray et al., Nature Biotechnology, 1993, vol. 11, pp. 64-70; Cottingham et al., Nature Biotechnology, 2001, vol. 19, pp. 974-977; Walsh et al., Nature Biotechnology, 2006, vol. 24, pp. 1241-1252; U.S. Patent Application Publication No. 2008/0167231.

  The peptides of the present invention may contain certain intermediate linkers useful for conjugating the peptide to the PEG moiety. Such linkers can provide minimal immunogenicity and toxicity to the host. Examples of such linkers are described in Bailon et al., PSTT, 1998, vol. 1 (8), pp. 352-356.

In certain embodiments, the present invention binds to a cysteine residue at position 28 of the amino acid sequence of SEQ ID NO: 29 with an isolated peptide consisting essentially of the sequence shown in SEQ ID NO: 29 containing CONH ₂ at the carboxy terminus. Intended for conjugation, including an intermediate linker. In certain embodiments, the intermediate linker is N-ethylsuccinimide. In a further embodiment, the intermediate linker is vinyl sulfone. In a further embodiment, the intermediate linker is acetamide. In a further embodiment, the intermediate linker is orthopyridyl disulfide.

In a further embodiment, the present invention comprises a peptide having the amino acid sequence shown in SEQ ID NO: 29 with CONH ₂ at the carboxy terminus, and an N-ethylsuccinimide linker linked to a cysteine residue at position 28 of SEQ ID NO: 29. For purposes of attachment, including where the N-ethylsuccinimide linker is also attached to the PEG moiety. In certain embodiments, the molecular weight of the PEG moiety can range from about 2 kDa to about 80 kDa. In certain embodiments, the mass of PEG is about 20 kDa. In a preferred embodiment, the CRHR2 peptide agonist comprises the peptide of SEQ ID NO: 82 or SEQ ID NO: 102. In certain embodiments, the PEG mass is about 5 kDa. In certain other embodiments, the PEG mass is about 12 kDa. In certain embodiments, the PEG mass is about 20 kDa. In certain embodiments, PEG has a mass of about 30 kDa. In certain embodiments, the PEG mass is about 40 kDa. In certain embodiments, the PEG mass is about 80 kDa. In certain embodiments, the PEG moiety is linear. In other embodiments, the PEG moiety is branched. The PEG moiety can be synthesized according to methods known to those skilled in the art. Alternatively, the PEG moiety is commercially available, eg, SUNBRIGHT® ME-020MA, SUNBRIGHT® ME-050MA, and SUNBRIGHT® ME-200MA (NOF corp. (Japan); Sigma Aldrich (St. Louis, MO, USA).

  The invention further relates to pharmaceutically acceptable salts of the peptides of the invention and methods of using the salts. A pharmaceutically acceptable salt refers to a free acid or base salt of a peptide that is non-toxic, biologically acceptable, or biologically suitable for administration to a subject in another manner. In general, S.M. M.M. Berge et al., “Pharmaceutical Salts”, J. Am. Pharm. Sci. 1977, 66: 1-19, and Handbook of Pharmaceutical Salts, Properties, Selection, and Use, Ed. Stahl and Wermuth, Wiley-VCH and VHCA, Zurich, 2002. Suitable pharmaceutically acceptable salts are pharmaceutically acceptable salts suitable for contacting a patient's tissue without causing excessive toxicity, irritation or allergic reaction. Examples of pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrroline Acid salt, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, sulphate Acid salt, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-diacid, hexyne-1,6-diacid, benzoate , Chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoic acid, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate ,citric acid , Lactate, γ-hydroxybutyrate, glycolate, tartrate, methane-sulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate and mandelate .

  When the peptide of the present invention contains basic nitrogen, any suitable method available in the art, for example free base is converted to an inorganic acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid, perchloric acid. , Sulfuric acid, sulfamic acid, nitric acid, boric acid, phosphoric acid, or organic acids such as acetic acid, trifluoroacetic acid, phenylacetic acid, propionic acid, stearic acid, lactic acid, ascorbic acid, maleic acid, hydroxymaleic acid, malic acid , Pamoic acid, isethionic acid, succinic acid, valeric acid, fumaric acid, saccharic acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, oleic acid, palmitic acid, lauric acid, pyranosidic acid such as glucuronic acid or galacturon Acids, α-hydroxy acids such as mandelic acid, citric acid or tartaric acid, amino acids such as asparagine Aromatic acids such as acid or glutamic acid, such as benzoic acid, 2-acetoxybenzoic acid, naphthoic acid or cinnamic acid, sulfonic acids such as laurylsulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, In light of any compatible mixture of acids such as methane sulfonic acid, ethane sulfonic acid, hydroxyethane sulfonic acid, cyclohexane sulfonic acid, acids exemplified herein and the ordinary level of skill in the art Desired pharmaceutically acceptable salts can be prepared, such as by treatment with any of the other acids considered to be equivalent or acceptable substitutes and mixtures thereof.

  Where the peptides of the invention contain acid groups such as carboxylic acids or sulfonic acids, any suitable method such as free acids can be converted to inorganic or organic bases such as amines (primary, secondary or tertiary). ), Any compatible mixture of bases, such as alkali metal hydroxides, alkaline earth metal hydroxides, bases exemplified herein, and the like, to the ordinary level of skill in the art Desired pharmaceutically acceptable salts can be prepared, such as by treatment with any of the other bases and mixtures thereof that are considered equivalents or acceptable substitutes in the light of. Specific examples of suitable salts include amino acids such as glycine and arginine, such as ammonia, carbonate, bicarbonate, primary, secondary and tertiary amines and cyclic amines such as benzylamine, pyrrolidine, piperidine. Organic salts generated from morpholine, piperazine, and the like, and inorganic salts generated from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium. Representative organic or inorganic bases further include benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, and procaine.

  The invention also relates to pharmaceutically acceptable prodrugs of this compound and methods of treatment using such pharmaceutically acceptable prodrugs. The term “prodrug” refers to a precursor of a specified compound that becomes a compound in a chemical or physiological manner after administration to a subject, such as solvolysis or enzymatic cleavage that occurs in vivo, or under physiological conditions. means. A “pharmaceutically acceptable prodrug” is a prodrug that is non-toxic, biologically acceptable, or otherwise biologically suitable for administration to a subject. An illustrative operating procedure for the selection and preparation of suitable prodrug derivatives is described, for example, in “Design of Prodrugs”, H.M. Bundgaard, Elsevier, 1985.

  Exemplary prodrugs include amino acid residues covalently linked to the free amino, hydroxy, or carboxylic acid group of the compound via an amide or ester bond, or two or more (eg, 2-4). Examples thereof include compounds having a polypeptide chain of amino acid residues. Examples of amino acid residues include 20 naturally occurring amino acids (which are generally represented by three letter symbols) as well as 4-hydroxyproline, hydroxylysine, demosin, isodesomosin, 3-methylhistidine, norvaline, beta -Alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine and methionine sulfone are included.

Additional types of prodrugs can be made, for example, by derivatizing free carboxyl groups in the structure of the compound to amides or alkyl esters. Examples of amides include amides generated from ammonia, primary C _1-6 alkyl amines and secondary di (C _1-6 alkyl) amines. Secondary amines include 5- or 6-membered heterocycloalkyl or heteroaryl ring moieties. Examples of amides include amides generated from ammonia, _C1-3 alkyl primary amines and di ( _C1-2 alkyl) amines. Examples of esters of the present invention include C _1-7 alkyl, C _5-7 cycloalkyl, phenyl and phenyl (C _1-6 alkyl) esters. Suitable esters include methyl esters. Prodrugs are also described in Fleisher et al., Adv. Drug Delivery Rev. Following procedures such as those outlined in 1996, 19, 115-130, groups containing hemisuccinate, phosphate ester, dimethylaminoacetate, and phosphoryloxymethyloxycarbonyl may be used to derivatize the free hydroxy group. Can be prepared. Carbamate derivatives of hydroxy and amino groups can also produce prodrugs. Hydroxy carbonate derivatives, sulfonate esters and sulfate esters can also provide prodrugs. Alternatively, the hydroxy group may be derivatized with (acyloxy) -methyl and (acyloxy) -ethyl ether (this acyl group may be an alkyl ester, optionally substituted with one or more ether, amine or carboxylic acid functions. Or the acyl group is an amino acid ester as described above) is also effective to produce a prodrug. This type of prodrug is described in Greenwald et al., J Med Chem. 1996, 39, 10, 1938-40. Free amines can also be derivatized as amides, sulfonamides or phosphonamides. All such prodrug moieties may incorporate groups including ether, amine and carboxylic acid functions.

  The invention further relates to pharmaceutically active metabolites of compounds that can be used in the methods of the invention. “Pharmaceutically active metabolite” means a pharmaceutically active product of metabolism of a compound or salt thereof in the body. Determination of compound prodrugs and active metabolites can be performed using routine techniques known or available in the art. See, for example, Bertolini et al., J Med Chem. 1997, 40, 2011-2016; Shan et al., J Pharm Sci. 1997, 86 (7), 765-767; Bagshawe, Drug Dev Res. 1995, 34, 220-230; Bodor, Adv Drug Res. 1984, 13, 224-331; Bundgaard, Design of Prodrugs (Elsevier Press, 1985); and Larsen, Design and Application of Prodrug, Drug Design. thing.

Pharmaceutical Compositions In certain embodiments of the invention, the CRHR2 peptide agonist is used to formulate a pharmaceutical composition alone or in combination with one or more additional ingredients. The pharmaceutical composition contains an effective amount of at least one active agent according to the present invention.

In some embodiments, the pharmaceutical composition comprises a peptide having the amino acid sequence set forth in SEQ ID NO: 29, wherein the peptide comprises CONH ₂ at the carboxy terminus and further bound to a cysteine residue at position 28. N-ethylsuccinimide linker or acetamide linker, wherein the linker is also linked to the PEG moiety. A PEG moiety is classified by its molecular weight and physical properties (eg, linear or branched) and includes one or more linker moieties used to attach PEG to the peptide substrate. In certain preferred embodiments, the peptide comprises one or two linkers.

  In certain embodiments, a pharmaceutical composition comprising the PEG moiety can include a PEG moiety that can weigh in a range of about 2 kDa to about 80 kDa. In certain embodiments, the mass of the PEG moiety is about 2 kDa. In a further embodiment, the PEG mass is about 5 kDa. In certain embodiments, the PEG mass is about 12 kDa. In certain embodiments, the PEG mass is about 20 kDa. In certain embodiments, the PEG mass is about 30 kDa. In certain embodiments, the PEG mass is about 40 kDa. In certain embodiments, the PEG mass is about 80 kDa. Such a composition may further comprise a pharmaceutically acceptable excipient.

  The present disclosure further includes compositions (including pharmaceutical compositions) comprising a compound or derivative described herein and one or more pharmaceutically acceptable carriers, excipients, and diluents. provide. In certain embodiments of the invention, the composition may further comprise a small amount of a wetting or emulsifying agent, or a pH buffering agent. In one particular embodiment, the pharmaceutical composition is pharmaceutically acceptable for administration to humans. In certain embodiments, the pharmaceutical composition comprises a therapeutically or prophylactically effective amount of a compound or derivative described herein. The amount of a compound or derivative of the invention that is therapeutically or prophylactically effective can be determined by standard clinical techniques. Representative effective amounts are described in detail in the following sections. In certain embodiments of the invention, the composition may also include a stabilizer. A stabilizer is a compound that slows the rate of chemical degradation of the modified peptide of the composition. Suitable stabilizers include, but are not limited to, antioxidants (such as ascorbic acid), pH buffers, or salt buffers.

  The pharmaceutical composition can be in any form suitable for administration to a subject, particularly a human subject. In certain embodiments, the composition is in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, and sustained release formulation. The composition may further be a specific unit dosage form. Examples of unit dosage forms include tablets; caplets; capsules (eg, soft elastic gelatin capsules); cachets; lozenges; lozenges; dispersions; suppositories; ointments; Creams; casts; solutions; patches; aerosols (eg nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to patients (suspensions (eg aqueous or non-aqueous liquid suspensions) Oil-in-water emulsion or water-in-oil liquid emulsion), solutions, and elixirs); liquid dosage forms suitable for parenteral administration to subjects; liquid dosage forms suitable for parenteral administration to subjects Examples include, but are not limited to, sterile solids (eg, crystalline or amorphous solids) that can be reduced.

  In one particular embodiment, the subject is a mammal such as a cow, horse, sheep, pig, poultry, cat, dog, mouse, rat, rabbit, or guinea pig. In a preferred embodiment, the subject is a human. Preferably, the pharmaceutical composition is suitable for veterinary administration and / or administration to humans. In accordance with this embodiment, the term “pharmaceutically acceptable” means approved by a federal or state regulatory agency for use in animals, and more specifically for use in humans, or the United States Means that it is listed in the pharmacopoeia or other generally approved pharmacopoeia.

  Pharmaceutical carriers suitable for use in the composition are sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. In one particular embodiment, the oil is peanut oil, soybean oil, mineral oil, or sesame oil. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Further examples of suitable pharmaceutical carriers are known in the art, e.g. W. Martin by Remington's Pharmaceutical Sciences (1990) 18th ed. (Mack Publishing, Easton Pa.).

  Suitable excipients for use in this composition include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, Examples include dry skim milk, glycerol, propylene, glycol, water, and ethanol. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition depends on various factors well known in the art, including but not limited to the route of administration of the composition and the particular active ingredient. Not.

  In certain embodiments of the invention, the composition is an anhydrous composition. An anhydrous composition can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. A composition comprising a modified peptide having a primary amine or secondary amine is expected to be substantially in contact with moisture and / or humidity during manufacture, packaging, and / or storage. Is preferably an anhydride. An anhydrous composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions are preferably packaged using materials known to prevent exposure to water so that they can be included in suitable formulation kits. Examples of suitable packaging include, but are not limited to, sealed foils, plastics, unit dose containers (eg, vials), blister packs, and strip packs.

  A pharmaceutical composition comprising a compound or derivative described herein, or a pharmaceutically acceptable salt and solvate thereof, is formulated to be compatible with its intended route of administration. This formulation is preferably for subcutaneous administration, but for administration by other means such as inhalation or insufflation (via mouth or nose), intradermal, oral, buccal, parenteral, vaginal, or rectal. possible. Preferably, the composition is further formulated to provide improved chemical stability of the compound during storage and transportation. The formulation can be lyophilized or can be a liquid formulation.

  In one embodiment, the compound or derivative is formulated for intravenous administration. Intravenous formulations can include standard carriers such as saline solution. In another embodiment, the compound or derivative is formulated for injection. In a preferred embodiment, the compound or derivative is a sterile lyophilized formulation and is substantially free of contaminating cellular material, chemicals, viruses, or toxins. In one particular embodiment, the compound or derivative is formulated in a liquid form. In another particular embodiment, the injectable formulation is supplied in a sterile single dose container. In one particular embodiment, the injectable formulation is supplied in a sterile single dose container. This formulation may or may not contain additional preservatives. Liquid formulations can take the form of suspensions, solutions or emulsions in oily or aqueous solvents and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

Methods of Administration The compounds or derivatives described herein, or pharmaceutically acceptable salts thereof, are preferably administered as a component of a composition that optionally includes a pharmaceutically acceptable solvent. This compound or derivative is preferably administered subcutaneously. Another preferred method of administration is via intravenous injection or continuous intravenous infusion of the compound or derivative. Preferably, this administration achieves a stable plasma concentration due to slow systemic absorption and clearance of the compound or derivative, or maintains a plasma concentration level within a predetermined range of time.

  In certain embodiments, the compound or derivative is administered by any other convenient route, such as infusion or bolus injection, or absorption through epithelial or dermal mucosal coatings (eg, oral mucosa, rectum, and intestinal mucosa). . The methods of administration are parenteral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, by inhalation. Or topically, particularly but not limited to ear, nose, eye or skin administration. In many cases, administration results in the release of the compound or derivative into the blood.

  Such dosage forms may be tablets, capsules, sachets, dragees, powders, granules, lozenges, powders for reconstitution, liquid preparations or suppositories. Preferably, the composition is formulated for intravenous or bolus injection, subcutaneous injection or bolus injection, or intramuscular injection.

  The compound is preferably administered by a parenteral route. For example, the composition may be constructed as a suppository for rectal administration. For parenteral use, including intravenous, intramuscular, intraperitoneal or subcutaneous routes, a sterile aqueous solution or suspension containing the agent of the present invention in a buffer to ensure proper pH and isotonicity Alternatively, it may be provided as a parenterally acceptable oil. Preferred aqueous solvents include Ringer's solution, dextrose solution, and isotonic sodium chloride. Such forms can be used for the purpose of producing unit dose forms such as ampoules or disposable injection devices, multiple unit dose forms such as vials from which appropriate doses can be removed, or injectable formulations May be provided in the form of a solid form or a preconcentrated liquid. Exemplary infusion doses may be administered over a period ranging from minutes to days. In yet another embodiment, an effective amount of a peptide of the invention can be coated onto nanoparticles suitable for subcutaneous delivery, or provided within a “depot” (Hawkins et al., Adv Drug Deliv Rev., 2008, vol. 60, pp. 876-885; Montalvo et al., Nanotechnology, 2008, vol. 19, pp. 1-7).

  The active agent can be administered through an inhalation procedure. Such methods include dry powder (Johnson KA, Adv Drug Del Rev., 1997, vol. 26 (1), pp. 3-15) and / or aerosol (Sangwan et al., J Aerosol Med., 2001). , Vol.14 (2), pp.185-195; PCT International Patent Publication No. WO2002 / 094342).

  In an embodiment of the method of treatment according to the invention, the therapeutically effective amount of at least one active agent according to the invention is a subject suffering from a disease, disorder or medical condition such as heart failure, diabetes, skeletal muscle consumption, and muscle loss. Or administered to a subject diagnosed with them. Additional conditions may include inappropriate athletic activity, dietary intake, or the need for cardioprotection, bronchial relaxation, and / or anti-inflammatory activity. Therapeutically effective amounts or doses of the active agents of the invention can be determined by routine methods such as modeling, dose escalation or clinical trials, and routine factors such as mode of administration or route or drug delivery, drug pharmacokinetics, disease, disease, And the severity and course of the condition, the treatment that the subject has received before or at present, the health status of the subject and the response to the medication and the judgment of the treating physician, etc. Typical intravenous administration rates range from about 0.2 ng to about 52 ng of stress copine-related active agents per minute per kg body weight of the subject, preferably from about 0.2 ng / kg / min to about 22 ng / kg. / Min, or equivalently, from about 0.3 μg / kg to about 32 μg / kg per day. In the case of bolus injection or subcutaneous injection, the total dose can be administered in a single dose unit or in divided dose units (eg BID, TID, QID, twice weekly, once every two weeks, or monthly Times). For a human weighing 70 kg, a typical range of suitable doses is from about 1 μg / day to about 1 mg / day. Instead of daily administration, a weekly dosage regimen can be used. In another preferred embodiment, the CRHR2 peptide agonist of SEQ ID NO: 102 comprising an acetamide linker in which about 20 kDa PEG is attached to a cysteine residue at position 28 of the peptide sequence is administered at 10 μg / kg by bolus subcutaneous injection. Dosage is administered to patients in need thereof. The frequency of this dose should range from once a day to less frequently, depending on the subject's therapeutic needs and other clinical considerations.

  Once improvement of the patient's disease, disorder or condition has occurred, the dosage may be adjusted to an amount suitable for prophylactic or maintenance treatment. For example, the dose and / or frequency of administration may be reduced to the extent that the desired therapeutic or prophylactic effect is maintained as a function of symptoms. Treatment may be stopped once symptoms have improved to an appropriate level. However, patients need intermittent treatment on a long-term basis when some symptoms recur.

  In certain embodiments, the compound is administered in combination with one or more other biologically active agents as part of a treatment regimen. In certain embodiments, the compound is administered before, simultaneously with, or after administration of one or more other biologically active agents. In one embodiment, one or more other biologically active agents are administered within the same pharmaceutical composition comprising a compound described herein. In another embodiment, one or more other biologically active agents are administered in a pharmaceutical composition that is separate from the compounds described herein. In accordance with this embodiment, one or more other biologically active agents can be administered to the subject by the same route as the route of administration used to administer the compound or by a different route.

  In another embodiment, the compound is administered with one or more other compounds or compositions for risk reduction or treatment of cardiovascular disease. Compounds or compositions that reduce or treat cardiovascular disease risk include anti-inflammatory agents, antithrombotic agents, antiplatelet agents, fibrinolytic agents, thrombolytic agents, lipid reducing agents, thrombin direct inhibitors, anti-Xa inhibitors Agents, anti-IIa inhibitors, glycoprotein IIb / IIIa receptor inhibitors and thrombin direct inhibitors, but are not limited to these. Examples of agents that can be administered in combination with the compounds described herein include bivalirudin, hirudin, hirugen, Angiomax, agatroban, PPACK, thrombin aptamer, aspirin, GPIlb / IIIa inhibitor (eg, Integrelin), P2Y12 inhibition Agents, thienopyridine, ticlopidine, and clopidogrel.

  In embodiments, the compound is formulated into a dosage form suitable for administration to a patient in need. For processes and equipment for the preparation of drug and carrier particles, see Pharmaceutical Sciences, Remington, 17th Ed. , Pp. 1585-1594 (1985); Chemical Engineers Handbook, Perry, 6th Ed. , Pp. 21-13-21-19 (1984); Journal of Pharmaceutical Sciences, Parrot, Vol. 61, no. 6, pp. 813-829 (1974); and Chemical Engineer, Hixon, pp. 94-103 (1990).

  The amount of compound incorporated into the dosage forms of the invention will generally range from about 10% to about 90% by weight based on the weight of the composition, depending on the therapeutic indication and the desired period of administration (eg, every 12 hours, every 24 hours, etc.). % Can vary. Depending on the dose of the compound desired to be administered, it can be administered in one or more dosage forms. Depending on the formulation, the compound is preferably in the HCl salt form or the free base form.

  Furthermore, the present invention also relates to a pharmaceutical composition or pharmaceutical dosage form as described above for use in a method of treatment or diagnosis of a human or non-human animal body.

  The present invention further relates to a pharmaceutical composition for use in the manufacture of a pharmaceutical dosage form for oral administration to a mammal in need of treatment, wherein the dosage form is independent of daily consumption by the mammal. It can be administered at any time.

  The present invention relates to a method of treatment or diagnosis of a human or non-human animal body, which comprises administering to the animal body a therapeutically or diagnostically effective amount of a pharmaceutical composition described herein. .

  The invention further relates to a pharmaceutical package suitable for commercial sale comprising the container, dosage form described herein, whether the dosage form can be administered with or without a meal, etc. With non-limiting documentation.

  The following formulation examples are limited for illustrative purposes and are not intended to limit the scope of the invention in any way.

Example 1: Peptide Synthesis and Purification The peptide of SEQ ID NO: 29 was prepared by solid phase peptide synthesis reaction using software version 3.3.0 on a Rainin Symphony multiple peptide synthesizer (model SMPS-110). It was. Resin used for peptide amide synthesis (NovaSyn TGR®, 440 mg, about 0.1 mmole, 0.23 mmol / g substitution, lot No. A33379) functions with acid labile modified Rink amide linker It was a composite of added polyethylene glycol and polystyrene.

  The amino acids used in the synthesis contained a Nα-9-fluorenylmethoxycarbonyl (Fmoc) protecting group at the C-terminus and the following side chain protecting group: Arg (2,2,4,6,7- Pentamethyldihydrobenzofuran-5-sulfonyl, pbf), Asp (tertiary butoxy, OtBu), Asn (trityl, Trt), Gln (Trt), Cys (Trt), His (Trt), Lys (t-butoxycarbonyl) , Boc), Ser (tertiary butyl, tBu) and Thr (tBu).

  The conjugation reaction was performed as follows: N-methylpyrrolidinone (NMP) swollen resin (0.1 mmole), 5-fold molar excess of Fmoc amino acid and 5-fold molar excess of hexafluorophosphorus in DMF (2.5 mL). Acid (HBTU) was mixed and a 10-fold molar excess of N-methylmorpholine (NMM) in DMF (2.5 mL) was added prior to binding for 45 minutes. The reaction was incubated with 20% piperidine / DMF solution for 2 minutes for Fmoc removal. The solution was drained and fresh 20% piperidine / DMF was added and incubated for 18 minutes. The reaction was then washed with NMP, followed by amino acid addition by repeating the coupling step. To join the C-terminus to Ile40, Gln39, Asn19, Asn12, and Val9 with numbers from the N-terminus, the coupling step was performed twice.

  Peptide cleavage from the resin was performed using trifluoroacetic acid (TFA) (100 mL), 1,2-ethanedithiol (EDT) (20.0 mL), phenol (7.5 g), thioanisole (5 mL), triisopropylsilane (TIS). ) (5 mL) and water (5 mL) and 9 mL of the cleavage mixture was performed using a 2 hour cleavage program and incubation. The cleaved peptide solution was transferred to a 50 mL BD polypropylene centrifuge tube and the peptide was precipitated with cold ethyl ether (40 mL). The mixture was centrifuged and ethyl ether was decanted from the peptide. Ethyl ether (40 mL) was added and the mixture was vortexed, centrifuged and decanted of ethyl ether. These steps (addition of fresh ethyl ether, vortex mixing, centrifugation, and decanting) were repeated two more times. The peptide was dried under reduced pressure to give 408 mg (92% yield) of crude product.

Peptide purification was performed on a Waters preparative HPLC system (Waters (MA, USA)). The crude peptide (approximately 100 mg) was dissolved in 20/30/50 acetic acid / acetonitrile / water containing 0.1% TFA. This material was injected into two Vydac C-18 columns (10 mm, 2.5 × 25 cm). After injection, the peptide was prepared using a gradient of 0-45% solvent B (solvent B = 80% acetonitrile with 0.1% TFA) for 5 minutes, 45-70% solvent B for 60 minutes using a flow rate of 6 mL / min. Purified. Fractions were collected and analyzed by analytical RP-HPLC, MALDI-TOF MS, and CE. The purest fractions were pooled and lyophilized to give 23 mg of product. MALDI-TOF MS measured a product molecular weight equal to 4400.5, which was one hydrogen atom greater than the molecular weight 4399.2 calculated for C ₁₉₅ H ₃₂₆ N ₅₆ O ₅₃ S ₃ . This liquid was freeze-dried by rapid freezing in an acetone dry ice bath for about 30 minutes. After freezing, the product in the open flask was covered with filter paper and placed under high vacuum. After 24 hours under high vacuum, the dried sample was removed from the vacuum and the storage container was sealed for later use.

Example 2: Conjugation of peptide and N-ethylmaleimide As shown in Scheme 1, N-ethylmaleimide capping on a site-derived cysteine residue was achieved under the following conditions.

  In a 2.5 mL polypropylene vial, 2.0 mg of the peptide of the present invention was dissolved in 1.0 mL of water. Next, 20 μL of 0.1 M N-ethylmaleimide aqueous solution was immediately added. The reaction was gently stirred at room temperature for 2 hours. This reaction mixture was subjected to a Summit APS (Dionex (CA, USA) equipped with a Vydac C18 10 × 250 mm (300 Å; Grace Davison (IL, USA)) column using the protocol shown in Table 6. A.)) Purified by HPLC. The terminal fractions were collected and analyzed by HPLC, and the pure fractions were accumulated and lyophilized.

Example 3 Conjugation of Peptide with Iodoacetamide-PEG Iodoacetamide-PEG (a linear 20 kDa polyethylene glycol chain with iodoacetamide ends, present in a limited amount at weakly alkaline pH, ) Resulted in cysteine modification as an exclusive reaction shown in Scheme 2. Cysteine thiol served as a selectivity point for the binding of iodoacetamide-PEG. The resulting derivative α-sulfahydrylacetamide linkage was achiral.

  To a 15 mL Erlenmeyer flask, 25 mg (5.68 mmol, 1.0 equiv) of the peptide of SEQ ID NO: 1 was added. To the same flask was added 140 mg (6.82 mmol, 1.2 eq, 95% active) PEG-20 iodoacetamide (Lot No. M77592) (manufactured by Nippon, Oil and Fat (NOF) Corp.). 10 mL of water was added and the solution was vortexed until all solids were dissolved. To this cloudy solution was added 50 mL of pyridine at a solution pH of about 8.91. After 2 hours, an aliquot of a 20 mL sample was removed and analyzed by reverse phase HPLC using a Phenomenex C6-phenyl column and 0.1% TFA / acetonitrile as the eluent. The sample showed that the reaction was almost complete after 2 hours (FIG. 1A). The reaction mixture was directly purified by HPLC using a Phenomenex C6 phenyl 10 × 150 mm column. The eluents for purification were 0.1% TFA water and 80% acetonitrile in 0.1% TFA water. Purification was performed in 2-3 mL sample batches (FIG. 1B). The purified fractions were combined and lyophilized in a 50 mL Erlenmeyer flask. The lyophilized solid was diluted with 10 mL water and lyophilized again. Approximately 1 mg of final product was diluted to 1 mg / mL and subjected to mass spectral analysis (FIG. 1C). The average weight of the PEGylated compound of SEQ ID NO: 102 was 25,449 daltons, partially due to the heterogeneity of the length of the PEG polymer, and the compound resulted as a white amorphous solid.

Example 4: PEGylation of peptide with N-ethylmaleimide linker In a 2.5 mL polypropylene vial, 2.0 mg (about 0.44 nmol) of peptide was dissolved in 2.5 mL of water and the amounts shown in Table 7 Then, immediately, activated N-ethylmaleimide-derived polyethylene glycols of various molecular weights were added.

  The reaction mixture was gently stirred at room temperature for 2 hours.

  This reaction mixture was subjected to a Summit APS (Dionex (CA, U.S.)) equipped with a Gemini 5u C6-phenyl 10 × 100 mm (110 Å; Phenomenex (CA, USA)) column using the protocol shown in Table 8. S.A.)) Purified by HPLC.

Example 5: CRHR2 and CRHR1 agonist activity-cAMP assay The CRHR2 and CRHR1 agonist activity of the CRH group was determined for SK-N-MC (human neuroblastoma) transfected with either human CRHR2 or human CRHR1. Two cell lines were characterized by adenosine 3 ′, 5′-cyclic monophosphate (cAMP) assay. h-SCP (SEQ ID NO: 1) has the same potency as h-UCN2 (SEQ ID NO: 115) in this assay and was shown to be the most selective CRHR2 agonist in the CRH group (Figure 2). ). The concentration required for 50% of maximum effect (A ₅₀ ) was 0.4 nM.

Human CRHR1 (accession number X72304) or CRHR2 (accession number U34587) was cloned into the pcDNA3.1 / Zeo expression vector and stably transfected into SK-N-MC cells by electroporation. Cells were 10% FBS, 50I. U. Retained in MEM with Earl's Salt containing 50 μg / mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate and 0.1 mM non-essential amino acids, 600 μg / mL G418 . Cells were grown at 37 ° C. in 5% CO ₂ .

  The cells were placed in a 96-well tissue culture dish (Biocoat, BD Biosciences) at 50,000 cells / well and cultured overnight. Cells were washed with PBS and then resuspended in DMEM F-12 without phenol red and with 10 μM isobutylmethylxanthine (IBMX). Cells were incubated for 60 minutes at 37 ° C. with peptides in a concentration range of 1 pM to 10 μM. Subsequently, to assess any antagonizing activity of the peptides that did not produce an agonist response, the peptides were preincubated for 20 minutes at 10 μM, then h-SCP was added and incubated for 60 minutes. Forskolin (10 μM) (a direct stimulator of adenylate cyclase) was used as a positive control. The assay was stopped by adding 0.5 M HCl and mixed by orbital rotation at 4 ° C. for 2 hours.

  In order to evaluate the activity of the peptides of the present invention on CRHR2, an intracellular cAMP assay using a Flash plate radioassay (Cat. No. Cus56088; Perkin Elmer (MA, USA)) was employed.

  Transfected SK-N-MC cells were placed in a 96-well Biocoat tissue culture dish (BD Biosciences (San Jose, CA, USA)) at 50,000 cells / well and cultured overnight. Cells were first washed with PBS and then suspended in DMEM / F-12 without phenol red and with 10 μM isobutylmethylxanthine (IBMX). The suspended cells were transferred to a 96 well flash plate coated with scintillant fluid. Cells were incubated for 60 minutes at 37 ° C. with peptides ranging from 1 pM to 1 μM. 10 μM forskolin was used as a positive control. After ligand stimulation, 0.5M HCl was added to lyse the cells and mixed by orbital rotation at 4 ° C. for 2 hours to release intracellular cAMP into the medium.

The medium containing the released intracellular cAMP was transferred to a 96-well flash plate coated with a scintillant solution containing an anti-cAMP antibody. In this assay, intracellular cAMP competes with ¹²⁵ I-labeled cAMP for binding to the antibody. In order to generate a standard curve, cAMP in the range of 2.5-250 pmol / mL was included in this experiment. [ ¹²⁵ I] -cAMP was measured with a TopCount scintillation counter (Perkin Elmer (MA, USA)).

Individual agonist concentration-response curve data is fitted to the Hill equation below using GraphPad Prism (Graphpad Software (La Jolla, CA, USA)) to produce half the maximum response. Provided an estimate of each parameter of the agonist concentration (A ₅₀ ), the maximum asymptote (α) and the Hill slope (n _H ) required for. In this equation, [A] is the agonist concentration and E is the measured effect:

For display purposes, the average fit parameter estimate was used to generate a single E / [A] curve shown superimposed on the average experimental data. The agonist efficacy estimate pA ₅₀ is expressed as the negative logarithm of the midpoint of each curve and is listed with the standard error of measurement (SEM). The base 10 logarithm (Log DR) value of the agonist dose ratio was calculated by subtracting the test compound pA ₅₀ value from the corresponding h-SCP (SEQ ID NO: 1) control pA ₅₀ value within the same assay batch. . The SEM value of the Log DR value is obtained by the square root of the sum of the squared SEM values of the h-SCP (SEQ ID NO: 1) control and test compound pA ₅₀ values.

The CRHR2-mediated cAMP response to h-SCP (SEQ ID NO: 1) is a concentration-dependent condition consistent with an overcoming competitive antagonistic activity, with a selective CRHR2 antagonist, anti-sorbazine-30 (SV30, SEQ ID NO: shown in Table 9). 118) (FIG. 3). The presence of anti-sorbazine-30 gave a pA ₂ value of 7.82 for the compound of SEQ ID NO: 1.

In this cAMP flashplate assay, human and rat peptides (see Table 10) were used to stimulate h-CRHR1 or h-CRHR2 transfected SK-N-MC cells. The peptide was incubated at 37 ° C. for 1 hour. Curves were calculated using a non-linear regression sigmoidal concentration response analysis calculation in GraphPad Prism. The pA ₅₀ values thus obtained are shown in Table 11 together with literature values.

斜体のデータは、有効性近似値を示す；ＮＡ＝低有効性及び限定的なペプチド供給量によりデータなし；論文データからの値は、下記の形質移入系のｃＡＭＰ刺激に使用された、執筆者の研究室内で合成されたペプチドで得られた：
¹ｈ−ＣＲＨＲ１又は² ｍ−ＣＲＨＲ２ｂ形質移入ＣＨＯ−Ｋ１細胞（Ｌｅｗｉｓ，Ｋ．ら、２００１，ＰＮＡＳ，ｖｏｌ．９８，ｐｐ．７５７０〜５）；
³ｈ−ＣＲＨＲ１又は⁴ ｈ−ＣＲＨＲ２ｂ形質移入ＨＥＫ−２９３細胞、濃度反応曲線から近似された値（Ｈｓｕ，Ｓ．Ｙ．ら、２００１，Ｎａｔ．Ｍｅｄ．，ｖｏｌ．７，ｐｐ．６０５〜１１）；
⁵ｍ−ＣＲＨＲ２ｂ形質移入されたＨＥＫ−２９３細胞（Ｂｒａｕｎｓ，Ｏ．ら、２００２，Ｐｅｐｔｉｄｅｓ，ｖｏｌ．２３，ｐｐ．８８１〜８８８）。

Italic data show efficacy approximations; NA = no data due to low efficacy and limited peptide supply; values from article data are for authors used for cAMP stimulation of the following transfection systems Obtained with peptides synthesized in the laboratory:
¹ h-CRHR1 or ² m-CRHR2b transfected CHO-K1 cells (Lewis, K. et al., 2001, PNAS, vol. 98, pp. 7570-5);
³ h-CRHR1 or ⁴ h-CRHR2b transfected HEK-293 cells, values approximated from concentration response curves (Hsu, SY et al., 2001, Nat. Med., Vol. 7, pp. 605-11) ;
⁵ m-CRHR2b transfected HEK-293 cells (Brauns, O. et al., 2002, Peptides, vol. 23, pp. 881-888).

  Recombinant non-amidated peptide libraries are difficult to assay in CRHR2-transfected SK-N-MC cells, so the effect of amidation of the C-terminal region of h-SCP on agonist activity In terms of efficacy and / or intrinsic activity.

  In order to investigate the peptide agonist activity contribution of various amino acids, several modified peptides were synthesized starting with 1-7 deletions within the N-terminal sequence. Each peptide was dissolved in water at a stock concentration of 1 mM, divided into aliquots, and stored in Eppendorf tubes (catalog number 023364111) at -40 ° C. The peptide was thawed only once on the day of the experiment and further diluted with cAMP assay buffer.

In each experimental replicate, all peptides that produced cAMP in h-CRHR2-transfected SK-N-MC cells achieved a similar maximum response. However, since the maximal response to h-SCP (SEQ ID NO: 1) varied between daily replicates, the data was normalized to the maximum response to h-SCP obtained within each replicate. The data was then combined from three to five replicate experiments for final calculation of agonist concentration-effect curve parameters (FIG. 4). The pA ₅₀ values obtained are summarized in Table 12.

Although the maximum response was indistinguishable, non-amidated h-SCP (SEQ ID NO: 113) was about 200 times more effective than the amidated parent peptide. In one batch, the parent 40 amino h-SCP peptide (SEQ ID NO: 1) is pA ₅₀ value was 9.41 ± 0.03. Terminal amidation is important but not essential for efficacy, and a fully defined concentration-effect curve was obtained with the non-amidated peptide with the same maximum response as the amidated parent peptide .

One amino acid deletion (SEQ ID NO: 107) had no significant effect on efficacy (pA ₅₀ 9.24 ± 0.05), three (SEQ ID NO: 108) and four (SEQ ID NO: 109) amino acid deletions) resulted in a decrease in each progressively pA ₅₀ values (8.49 ± 0.08 and 7.33 ± 0.9). This is shown in Table 12. Five or more amino acid deletions (SEQ ID NO: 110, SEQ ID NO: 111 and SEQ ID NO: 112) completely abolished agonist activity (FIG. 4). Therefore, the latter three peptides were tested as antagonists of h-SCP at a concentration of 10 μM (FIG. 5). None of the peptides had a significant effect on the h-SCP concentration-effect curve. This indicates that these peptides not only have no detectable intrinsic efficacy, but also have no significant receptor occupancy (ie, an affinity of less than 10 μM).

Deletion of 4 or more amino acids in the N-terminal region of the h-SCP sequence affects peptide efficacy. Peptides with 1-4 amino acid deletions in the N-terminal region are progressively reduced in effectiveness, whereas peptides with 5 or more amino acid deletions result in complete loss of agonist activity and receptor affinity. (K _A > 10 μM). The latter was predicted based on previous reports of similar analyzes performed on h-UCN2 (Isfort, RJ et al., 2006, Peptides, vol. 27, pp. 1806-1813). . This is because the deletion is close to the retained position 6 amino acid serine and position 8 aspartic acid.

ＮＲ＝反応なし

NR = no response

In addition, the effects of cysteine mutation, N-ethylmaleimide capping, and PEGylation on peptide agonist activity were investigated. control pA ₅₀ of h-SCP (SEQ ID NO: 1) is 9.47 to 9.74 in various assay batches, SEM varies with from 0.03 to 0.11. Again, some modified peptides were synthesized according to the above scheme. The assay results for these peptides are shown in Table 13.

  Results illustrating the activity profiles of various modifications of the peptides of the present invention are shown in Table 14. This includes the stress copine (h-SCP) peptide, urocortin 2 (h-UCN2), and h-SCP-IA-PEG peptide (SEQ ID NO: 102), where h-SCP-IA-PEG has the sequence It is a peptide having an SCP sequence with cysteine substitution at position 28 represented by number 29, and a PEG polymer linked to the cysteine at position 28 via an acetamide (IA) linker. This data is the mean ± SEM of 1 to 3 replicates and is expressed as the% of the maximum response obtained for h-SCP within each replicate experiment.

h-SCP-IA-PEG peptide was also incubated in the presence of 100 nM anti-sorbazine-30 (a selective competitive antagonist of h-CRHR2 receptor), and in the h-SCP-IA-PEG peptide concentration-response curve The corresponding approximate pA ₅₀ value was 6.89 when it resulted in a shift to the right and the maximal response was constrained to 100%.

Example 6: Radioligand binding activity of CRHR1 and CRHR2 The binding profile of h-SCP (SEQ ID NO: 1) with CRHR2 is stably transfected with human CRHR2 using [ ¹²⁵ I] -antisorbazine-30 as a radiolabel. Determined in radioligand binding studies in membrane preparation of cultured SK-N-MC cells. The cells were harvested by cell scraping and the resulting pellet was immediately frozen at −80 ° C. (approximately 50 × 10 ⁶ cells / pellet).

The frozen cell pellet was thawed on an ice bath in 15 mL assay buffer. This buffer consisted of 10 mM HEPES, 130 mM NaCl, 4.7 mM KCl, 5 mM MgCl ₂ , and 0.089 mM bacitracin, pH 7.2, and 21 ± 3 ° C. This solution was homogenized using a Polytron tissue grinder with settings 10 and 7 × 3 s (Brinkmann Instruments (Westbury, NY)). The homogenized solution was centrifuged at 800 × g for 5 minutes at 4 ° C., and the pellet was discarded. This supernatant was again centrifuged at 26,892 × g for 25 minutes at 4 ° C., and the final pellet was resuspended in assay buffer. All binding assays were performed in 96-well Multiscreen GF / B filter plates (Millipore (Billercay, MA, USA)) that had been pre-soaked for 1 hour in assay buffer containing 0.3% PEI. It was done. To complete the study, a volume of 45 μL of cell membrane was added with 60 pM [ ¹²⁵ I] -antisorbazine-30 in a volume of 50 μL for CRHR2 assay or with [ ¹²⁵ I]-(Tyr ⁰ ) -sorbazine for CRHR1 assay. , Incubated in the presence of 15 μL of competing ligand for a total volume of 150 μL for 120 minutes. Nonspecific binding was determined by including 1 μM r-UCN1 (SEQ ID NO: 114). The bound radioactivity was separated by filtration using a Multiscreen Resist manifold (Millipore Corp. (Billerica, MA, USA)). The filter was washed 3 times with ice-cold PBS at pH 7.5 and the radioactivity remaining on the filter was quantified by liquid scintillation as measured by a TopCount counter (Packard BioScience (Boston, MA, USA)). . All experiments were performed in triplicate.

Data from individual competition curves were expressed as a percentage of specific [ ¹²⁵ I] -anti-sorvazine-30 or [ ¹²⁵ I]-(Tyr ⁰ ) -sorbazine binding (B) within each experiment. These data are then compared with competitors using a 4-parameter logistic using GraphPad Prism, each with an upper asymptote (α _max ) weighted to 100% and a lower asymptote (α _min ) weighted to 0%, respectively. Were analyzed by including these values two log units above and below the lowest and highest concentrations of:

The competition curve obtained with h-SCP (SEQ ID NO: 1) was biphasic. This indicates a high affinity and low affinity receptor binding state characterized by a high negative logarithm of concentration at 50% inhibition (pIC ₅₀ ) and a low pIC ₅₀ of 6.6. It was. High affinity site binding has been shown to be inhibited by 100 μM guanosine 5′-O- [γ-thio] triphosphate (GTPγS). In contrast, h-UCN2 (SEQ ID NO: 115) exhibited only high affinity binding. This indicates that h-UCN2 is acting as an agonist with higher intrinsic efficacy than h-SCP (SEQ ID NO: 1) in the assay. The pK _I values obtained from this data analysis are shown in Table 15.

^*ＮＤ＝検出不可能

^* ND = not detectable

Example 7: Relaxation of vascular smooth muscle-rat aortic ring The ability of h-SCP (SEQ ID NO: 1) to relax vascular smooth muscle in isolated rat aortic rings pre-contracted with phenylephrine (PE) was examined (Fig. 6). This peptide (SEQ ID NO: 1) produced a concentration-dependent relaxation with a pA ₅₀ of 6.05 ± 0.12, but h-UCN2 (SEQ ID NO: 115) with a pA ₅₀ of 7.01 ± 0.13. ) Was 1/10 the effectiveness. The response elicited by h-SCP (SEQ ID NO: 1) was inhibited by anti-sorbazine-30 (SEQ ID NO: 118).

Example 8: Cardiovascular Characterization in Isolated Rabbit Hearts Effect of h-SCP (SEQ ID NO: 1) on heart rate (HR), left ventricular (LV) contraction, and vascular tone in retrograde perfusion Langendorff assay of rabbit heart Was evaluated. A placebo-like control vehicle or h-SCP (SEQ ID NO: 1) bolus was administered directly into the perfusion block. h-SCP (SEQ ID NO: 1) produces a concentration-dependent increase in heart rate, with the left ventricle increasing pressure (dP / dt _max ), 50% response concentration equal to 52 nM, 9.9 nM, and 46 nM, respectively. Coronary vascular perfusion pressure (CPP) decreased at the same time (FIG. 7), but no response was observed with the control solvent.

  While the foregoing specification teaches the principles of the invention, along with examples provided for purposes of illustration, the practice of the invention is all included within the scope of the following claims and their equivalents. It will be understood to encompass the usual variations, adaptations and / or modifications of

Claims (32)

A peptide having agonist activity against corticotropin releasing hormone receptor type 2, wherein the peptide has the amino acid sequence:
LSLDV PTNIM NLLFN IAKAK NLRAQ AAANA HLMAQ I
Wherein at least one amino acid of the peptide is substituted with X, provided that the substitution is not made at positions 3, 29, and 33 of the amino acid sequence, and where X is cysteine, tyrosine, or glutamic acid A peptide,
Or a pharmaceutically acceptable salt or amide thereof. Said substitution is
X instead of L at position 1;
X instead of S at position 2;
X instead of D at position 4;
X instead of V at position 5;
X instead of P at position 6;
X instead of T at position 7;
X instead of N at position 8;
X instead of I at position 9;
X instead of M at position 10;
X instead of N at position 11;
X instead of L at position 12;
X instead of L at position 13;
X instead of F at position 14;
X instead of N at position 15;
X instead of I at position 16;
X instead of A at position 17;
X instead of K at position 18;
X instead of A at position 19;
X instead of K at position 20;
X instead of N at position 21;
X instead of L at position 22;
X instead of R at position 23;
X instead of A at position 24;
X instead of Q at position 25;
X instead of A at position 26;
X instead of A at position 27;
X instead of A at position 28;
X instead of A at position 30;
X instead of H at position 31;
X instead of L at position 32;
X instead of A at position 34;
2. The peptide of claim 1 selected from the group consisting of X instead of Q at position 35; and X instead of I at position 36. The peptide is
XLSLD VPTNI MNLLF NIAKA KNLRA QAAAN AHLMA QI-NH ₂ ,
XTLSL DVPTN IMNLL FNIAK AKNLR AQAAA NAHLM AQI-NH ₂ ,
XFTLS LDVPT NIMNL LFNIA KAKNL RAQAA ANAHL MAQI-NH ₂ and XKFTL SLDVP TNIMN LLFNI AKAKN LRAQA ANAH LMAQ1-NH ₂   The amino acid sequence is selected from the group consisting of SEQ ID NOs: 17, 18, 20, 21, 25, 26, 27, 29, 32, 33, 34, 36, and 41; and X is cysteine. The peptide according to 1.   A complex comprising the peptide according to claim 1 and a linker bound to X of the peptide.   6. The complex according to claim 5, wherein X is cysteine.   6. The complex according to claim 5, wherein the linker is acetamide or N-ethylsuccinimide.   6. The complex of claim 5, further comprising polyethylene glycol (PEG) attached to the linker, wherein the PEG has a molecular weight of about 80 kDa or less.   9. The complex of claim 8, wherein the linker is acetamide.   9. The complex of claim 8, wherein the PEG has a molecular weight selected from the molecular weight group consisting of about 2 kDa, about 5 kDa, about 12 kDa, about 20 kDa, about 30 kDa, and about 40 kDa.   6. The complex of claim 5, wherein the PEG is branched or linear.   6. The complex of claim 5, wherein the PEG further comprises a reactive group.   The composite according to claim 12, wherein the reactive group is N-ethylmaleimide.   The peptide is SEQ ID NO: 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, The peptide of claim 1 having an amino acid sequence selected from the group consisting of 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 39, 40, and 41. formula:

Wherein R is the peptide of claim 4 and S is the sulfur atom of the cysteine thiol group of X. formula:

Where n is an integer ranging from about 40 to about 1900, and R is SEQ ID NO: 2, 3, 4, 5, 6, 10, 13, 16, 17, 18, 19, 20, 21, 22, 24, The peptide according to claim 1, which has an amino acid sequence selected from the group consisting of 25, 26, 27, 28, 29, 30, 32, 33, 35, 36, 37, 39, 40, and 41, and S Is a sulfur atom of a cysteine thiol group of X. formula:

The bPEG is a branched polyethylene glycol having a molecular weight of about 80 kDa, R is a peptide having the amino acid sequence of SEQ ID NO: 29, and S is a sulfur atom of the cysteine thiol group of X. Complex. formula:

Wherein n is an integer ranging from about 40 to about 1900, R is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 29, and 36, and S is the sulfur of the cysteine thiol group of X The composite according to claim 6, which is an atom.   The peptide of claim 1, wherein the peptide has the amino acid sequence of SEQ ID NO: 29.   17. The complex of claim 16, wherein n is an integer of about 460 and R is a peptide having the amino acid sequence of SEQ ID NO: 29.   19. The complex of claim 18, wherein n is an integer of about 460 and R is a peptide having the amino acid sequence of SEQ ID NO: 29.   A polynucleotide encoding the peptide of claim 1.   A pharmaceutical composition comprising (a) the peptide of claim 1 and (b) a pharmaceutically acceptable excipient.   24. The pharmaceutical composition according to claim 23, comprising a peptide having the amino acid sequence of SEQ ID NO: 29.   A pharmaceutical composition comprising (a) the complex of claim 5 and (b) a pharmaceutically acceptable excipient.   A pharmaceutical composition comprising (a) the complex of claim 20 and (b) a pharmaceutically acceptable excipient.   A pharmaceutical composition comprising (a) the complex of claim 21 and (b) a pharmaceutically acceptable excipient.   A monoclonal antibody that specifically binds to a peptide comprising the amino acid sequence of the peptide of claim 1.   The monoclonal antibody according to claim 28, wherein the peptide is PEGylated.   A corticotropin releasing hormone receptor type 2 activity selected from the group consisting of metabolic disease and heart failure comprising administering a therapeutically effective amount of the peptide of claim 1 to a subject in need of treatment. A method of treating a subject afflicted with, or diagnosed with, a mediated disease, disorder and medical condition.   32. The method of claim 30, wherein the disease, disorder, or medical condition is diabetes.   32. The method of claim 30, wherein the disease, disorder or medical condition is heart failure.

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